Relation between the risk factors for the severity of denture stomatitis and quality of life of complete edentulous individuals: a cross-sectional study

Abstract Objective To assess the association between risk factors for developing denture stomatitis (DS) and oral health-related quality of life (OHRQoL) in complete denture wearers. Methodology Participants of both sexes, wearing complete dentures, were classified using the modified Newton classification for the absence or the severity of DS and allocated to groups Normal or zero, IA, IB, II, and III. Lifestyle, oral and denture history, and medication use were assessed using specific questionnaires; clinical parameters such as anatomical characteristics of support were evaluated with the Kapur classification; salivary flow (SF) was calculated by the volume of unstimulated saliva per minute; and microbial load was determined by counting colony forming units (CFU) of target microorganisms present in the biofilm collected from dentures and palate. OHIP-EDENT assessed the OHRQoL. Kendall's tau_b and Spearman tests were applied with a significance level of 5%. Results 184 patients (143 female and 41 male) aged 65.5 ± 6.8 years were evaluated. Positive correlations were found for sex (women; p=0.013, r=0.16), individuals who started to consume alcoholic beverages as a young adult (18–27 years) (p=0.008, r=0.22), CFU of Candida spp. (p<0.001, r=0.27 denture; p<0.001, r=0.31 palate); Candida albicans (p=0.004, r=0.22 denture; p=0.003, r=0.25 palate), and Candida glabrata (p=0.004, r=0.22 denture; p=0.001, r=0.27 palate). Moreover, negative correlations with DS were found for CFU of Staphylococcus spp. (p=0.004, r=-0.20 palate) and enterobacteria (p=0.002, r=-0.24 palate), as well as a negative correlation between SF (p=0.009, r=-0.193) and DS. The CFU of Staphylococcus spp. and enterobacteria on the palate significantly correlated with OHRQoL. Conclusion Being female, consuming alcoholic beverages as a young adult, CFU of Candida spp., Candida albicans, Candida glabrata, and salivary flow may be the most significant risk factors for DS. The microbial load of Staphylococcus spp. and enterobacteria seems to influence the quality of life for complete denture wearers.


Introduction
Although treatment with complete dentures provides patients with a better quality of life, 1 these devices may be associated with oral lesions, the most common of which are denture stomatitis (DS), 2,3 angular cheilitis, and traumatic ulcers. 4DS is clinically characterized by different degrees of chronic inflammation of the tissue that supports the complete denture, usually located in the maxillary ridge and palate; [5][6][7][8] it can be asymptomatic or manifest with burning in the palate region, especially when swallowing. 9 The degree of DS can be diagnosed according to modified Newton's classification, by the attribution of scores. 7The modified Newton's classification introduces two subtypes for Newton type I, resulting in: type 0: healthy mucosa; type IA: petechiae in normal palatal tissue, usually found around the orifices of the ducts of the palatal mucous glands; type IB: localized area of inflammation in the denture-bearing area; type II: generalized area of inflammation in the denturebearing area; and type III: hyperplasic palatal surface with inflammation in the denture-bearing area. 7Among the etiological factors, local factors stand out, such as fungal infection, especially for Candida spp., 2,3,[10][11][12][13][14] biofilm accumulation on dentures, 3,13,14 poor denture hygiene, mucosal trauma due to adaptation problems, or the continuous and nocturnal use of dentures. 3,9stemic factors that affect the immune system can be cited as the cause of DS. 11, 15 Specifically, elderly patients with diabetes have been reported to be around 4.4 times more likely to develop denture stomatitis than a healthy patient. 14Patients who use prescription drugs such as antidepressants, antihypertensives, diuretics, or have other conditions related to xerostomia, have a higher prevalence of stomatitis associated with dentures. 14However, only a few factors have been statistically associated with DS, and it is essential to consider age, sex, age of dentures, and time of use to determine the possible risk factors for DS. 4,8,14,16Most patients positive for DS do not report specific symptoms of the disease, so they often remain undiagnosed and untreated for extended periods, 17 which can complicate their systemic health 18 and impact the quality of life. 19though the literature states that local and systemic factors are associated with the cause of DS, few studies have investigated, with clinical trials, the relationship between risk factors of DS and OHRQoL. 19erefore, the primary aim of this study was to evaluate the socioeconomic profile and assess the association between risk factors such as lifestyle, oral and denture history, clinical parameters, medication use, salivary flow, and microbial load parameters of completely edentulous patients who were healthy or with different severities of DS.The secondary aim was to correlate all the variables with OHRQoL.This information may contribute to enabling more appropriate management in the prevention and treatment of DS, as well as helping to maintain these individuals' oral and denture hygiene, especially in the promotion of alternatives aimed at public health procedures for this population composed mainly of the elderly.The null hypothesis tested was that socioeconomic profile, lifestyle, oral and denture history, clinical parameters, medication use, salivary flow, and microbial load would be similar for healthy patients and those with different severities of Denture stomatitis, and that these factors would not influence OHRQoL.The inclusion criteria were that the participants were of both sexes, completely edentulous, wore complete dentures, and were healthy or positive for different severities of DS, according to the modified Newton Classification. 7The exclusion criteria were individuals with dentures with adaptation problems, relining, repairs or fractures, and subjects with oral according to the methodology described by Kapur. 21e maxillary and mandibular ridge was assessed on a scoring scale from 1 to 4, with 1 being the most unfavorable clinical scenario and 4 the most favorable.

Methodology
The supporting tissues and muscle insertions were evaluated according to a scale of 1 to 3. The clinical score for each case was obtained by adding the values attributed to the maxilla and mandible, and the result (total) was assigned to the following conditions: unfavorable/bad clinical conditions (less than 14); satisfactory clinical conditions (between 14 and 17); and correct clinical conditions (higher than 17). 21All saliva samples were collected from 9:00 to 11:00 AM.The participants were asked to spit into a tube for 10 minutes to collect unstimulated total saliva.The volume of each patient's saliva was recorded to evaluate the salivary flow rate in mL/minute.
The denture biofilm was collected by desorption method with a sterile brush (Tek, Johnson & Johnson do Brasil Indústria e Comércio de Produtos para Saúde Ltda., SJ dos Campos, SP, Brazil) and buffer solution (PBS-phosphate buffered saline) in an aseptic zone. 22 collect the palate samples, a sterile cytology brush was rubbed in the palatal regions affected by DS for 1 minute. 7Then, the active tip was removed and stored in a sterile tube containing 5 mL of PBS solution.The solutions with the samples collected from the dentures and the palate were stirred for 1 minute with the aid of a Vortex shaker (Phoenix® -AP56, Ind. E Com. De Equip.Científicos Ltda, Araraquara, SP, Brazil).
Next, 50 μL of the solutions were diluted in 450 μL of PBS, obtaining serial dilutions of 10 -¹ to 10

Data analysis
The data showed a non-normal distribution
As for habits and lifestyle, negative correlations were identified with alcoholic drink consumption (r=-0.149;p= 0.006), and patients that never and rarely partake in alcoholic beverages presented higher degrees of DS.However, among patients who consumed alcoholic drinks, patients who started drinking alcohol when they were young adults (18-27 years) had a higher degree of DS (r=0.022,p=0.008) (Table 2).The medication frequency reports (Table 3) show that only patients who are users of antidepressants showed a negative correlation with the salivary flow rate (r=-0.129;p=0.038), and SF presented a significantly negative relation with DS (r=-0.194,p=0.009).
As for oral and denture history, these factors showed that patients that never visited dentists or visited dentists only in emergencies presented a higher degree of DS.Moreover, patients with complete mandibular dentures over six years old or more presented a positive correlation with DS, as shown in Table 4.The Kapur index did not correlate significantly with the degree of DS (r=0.114;p=0.07).
For the correlation between the microbial load and the degree of DS (Table 5) Enterobacteria were related to social disability, and discomfort and psychological disability (domains 3 and 4; respectively; r=0.25, p=0.001; r=0.17, p=0.02) of OHIP-EDENT.It is noteworthy that all factors were evaluated concerning quality of life.Only D1 showed no correlation with any of the variables assessed.

Discussion
Female, the onset of alcohol consumption in young adulthood (18-27 years), the presence of Candida spp., Candida albicans, and Candida glabrata on dentures and palate, and salivary flow were associated with denture stomatitis, and the null hypothesis was therefore rejected.
Among the sociodemographic data studied, there was a correlation only between sex and degree of DS.
The greater severity of DS was correlated with females.
The literature suggests that this relationship is due to attitudinal and physiological issues. 4ncerning attitude, women seek dental treatment more frequently than men, in addition to wearing their dentures for prolonged periods. 24However, the logic of this explanation is different from the achieved results.
Frequent visits to the dentist should reflect better care and guidance about oral health and a lower rate of DS. 24 Moreover, another fact that could be considered is that the sample was predominantly made up of women, which could lead to the hypothesis of a greater number of reported cases of denture stomatitis, opposite to what was observed in males, which could represent a situation of underreported cases of denture stomatitis.However, this epidemiological profile and its correlation with DS could be addressed in future studies to confirm or refute such hypotheses.
Regarding physiological issues, hormonal changes before and after menopause contribute to the atrophy of bone support, which causes chronic irritation in poorly adapted dentures. 4,25The women in this study were aged over 59 years; therefore, they were all post-menopausal, corroborating this hypothesis in the literature.It was expected that age, education level, and income would correlate with DS, but this was not observed in the results.Although correlated with DS, the information on marital status and "lives with" was intended to characterize the sample, and no significant correlation results were expected.
Regarding lifestyle, there was an unexpected correlation between never consuming alcoholic beverages and a higher degree of denture stomatitis.
Many patients (n=97, 52.7%) said they had never consumed alcohol.Although the number of patients is considerable, this relationship should be interpreted cautiously, since alcohol can be related to lesions in epithelial tissue. 26However, when assessing the patients who reported consuming alcoholic beverages (n=85, 46.2%), it was noted that the onset of alcohol consumption in young adulthood (18-

These examples of medical history and medication use represent a scenario of elderly patients commonly
reported in other studies. 27,28e patients who used a class of antidepressants It was observed that patients who went to the dentist only for emergencies or who never went had a positive correlation with a higher degree of DS.The patient's lack of visits to the dentist contributes to a lack of information regarding hygiene methods, which contributes to the severity of stomatitis.Also, in most patients DS can manifest asymptomatically, adding to the impossibility of self-diagnosis, and can establish itself for long periods without any diagnosis.In previous studies, a relationship between the frequency of visits to the dentist and DS was determined. 29garding denture history, it was observed that

Questions
Frequency ; Q2-How often do you see a dentist?; Q3-Presence of burning on the palate?; Q4-Presence of burning on the tongue?; Q5-How many years are you edentulous?; Q6-How old is your current maxillary denture?; Q7-How old is your current mandibular denture?0, IA, IB, II, III are degrees of DRS according to the Newton Modified Classification.Being, 0: healthy mucosa; IA: petechiae in normal palatal tissue; IB: localized area of inflammation of the denture-bearing area; II: generalized area of inflammation of the denture-bearing area; III: hyperplasic palatal surface with inflammations of the denture-bearing area.
Table 4-Descriptive and inferential analysis of the correlation of dental and oral history, and dentures of the participants with degree of DS Relation between the risk factors for the severity of denture stomatitis and quality of life of complete edentulous individuals: a cross-sectional study J Appl Oral Sci.
2023;31:e20230192 9/13 patients who had complete mandibular denture for over six years or more presented a positive correlation with DS.It was expected that this correlation would be with the maxillary denture, the region with the highest prevalence of mucosal lesions, 7 nonetheless, one possibility to justify the longer use of the mandibular denture and the higher degree of DS is a possible change in the vertical dimension of occlusion, an important factor that correlates with the trauma factor of DS.However, this study did not directly evaluate the vertical dimension of occlusion, but in the literature it is established that this factor may have a direct relationship with mucosal lesions in complete edentulous patients. 16ncerning anatomical features and their correlation with DS, to our knowledge, this is the first article to report these factors.Although   information for a favorable or unfavorable prognosis regarding the quality of the dentures, 21 which is directly related to the etiologies of mucosal epithelial lesions.
The sample for this study was composed of participants from the public health system in the city of Ribeirão Preto and the surrounding region, who sought care at the School of Dentistry of Ribeirão Preto for replacement dentures.They showed characteristics common to the South American edentulous population, 4,13,19,20 and the results showed that the main factor involved in the risks related to DS was the presence of biofilm rich in pathogenic microorganisms, possibly due to a failure of maintaining oral health and dentures.
The microorganisms Candida spp., enterobacteria, Staphylococcus spp., and S. mutans were selected and evaluated because they are species commonly found in the biofilm of complete dentures, 13,20 Candida albicans in particular being highly correlated with the presence of DS. 20,30 In this study, positive correlations were found between CFU of Candida spp., C. albicans, and C. glabrata (dentures and palate) and a higher degree of DS.The highly effective adhesion of these microorganisms to the denture acrylic base justifies this finding, which corroborates several studies already reported in the literature. 4,20,25This result confirms the need for special care with denture hygiene as a form of preventing DS, 31 since hygiene contributes to a reduction in biofilm and microbial load, promoting better contact between the inner surface of the dentures and the mucosa, which decreases the risk of worsening systemic conditions due to the opportunism of the microorganism. 8,16,17,20 A recent literature review 33 showed that when which would be an interesting assessment to address in future studies, particularly in patients who do not respond to appropriate initial therapy. 33r S. mutans, no correlation with DS was found.
This may be due to the fact that it is a microorganism related to the initial adhesion of microorganisms, being a precursor to biofilm formation. 12,20As most of the patients evaluated had been using their dentures for a long time, it is believed that the dentures deposits already presented a more complex structure of the biofilm collected.
Although the literature suggests that chronic DStype injuries can influence the quality of life, there is still no direct evidence of this relationship when considering denture stomatitis. 24According to the authors of this study, this is the second time that this relationship has been reported. 19,24However, for microbial load, which is one of the factors strongly related to DS, there is still no data on its relationship with quality of life.
Although Staphylococcus spp.showed a negative correlation with the degree of DS, the patients with a higher CFU of Staphylococcus spp.had a lower quality of life.It was expected that the microbial load of other microorganisms, especially Candida spp., would also correlate with the quality of life.However, it can be assumed that Staphylococcus spp.can coexist with other microorganisms, 33 potentiating severity and symptomatology, mainly on the palate, the region with the greatest manifestation of DS. 7 However, the microorganisms were evaluated separately in relation to quality of life.
Staphylococcus spp.showed a positive correlation with poor quality of life in three domains (D2: Psychological discomfort and disability; D3: Social disability; and D4: Oral pain and discomfort), enterobacteria with two domains (D3 and D4), and for the other microorganisms no significant correlation was observed between CFU and quality of life.This may be due to the fact that local chronic inflammation, mediated by microorganisms, is related to epithelial lesions that can trigger edematous ulcerated sites, with symptoms of pain and burning, commonly seen in DS, 7 which can interfere with the intimate adaptation of the dentures to the supporting tissue, and impact on patient satisfaction and quality of life. 19Additionally, the hypothesis that microorganism presence could promote injured tissue and, consequently, pain and discomfort, can be reinforced by the correlation between microbial load and quality of life. 19further problem was the domain of social disability, that is, the impact of oral conditions on social relationships.Those who experienced embarrassing situations and less tolerance from their spouse and family had a higher CFU of Staphylococcus spp.and enterobacteria.This result may be related to the microorganisms identified in the denture and palate, which promoted staining and odors, signs and symptoms that can cause discomfort and insecurity in these participants' social life. 19,24is study presented some limitations.One of these was not evaluating the quantity of biofilm on the two surfaces (denture and palate).However, the presence of a microbial load of all pathogens may suggest that biofilm was present on these surfaces.
Another area of limitation is the design of a crosssectional study without the power of a justified causal relationship between all the factors of DS and quality of life.Future research should clarify this relationship using a randomized and controlled study to clarify cause-effect relations. 16her limitations of this study are that the denture vertical occlusal dimension, retention, and stability were not evaluated to quantify them as possible risk factors.Only one clinical evaluation was carried out and did not include individuals with evident denture quality problems.However, these are necessary factors to consider in future studies.
Study populationThis cross-sectional study was approved by the ethics committee at the School of Dentistry of Ribeirão Preto, University of São Paulo (CAAE: 3712418.1.0000.5419).All participants gave fully informed written consent and were recruited from August 2018 to February 2023.
photographs of the palate of participants without the identifiable name or clinical information (C.H.S-L., H.F.O.P.).Standardized photographs of the palate -3 and added to Petri dishes with the following specific culture media: CHROMagar (CHROMagar™ Candida, Becton Dickinson, Paris, França) for isolation of Candida spp.; MacConkey Agar (Himedia Laboratories PVI Ltd, Mumbai, Índia) for isolation of Gram-negative microorganism; Manitol Agar Salt (Kasvi Imp. e Dist.de Prod.para Laboratórios Ltda, Curitiba, Brasil) for isolation of Staphylococcus spp.; and SB20 Agar Modified by casitone for isolation of Streptococcus mutans.The plates were incubated in a microbiological incubator (De Leo Equipamentos Laboratoriais, Porto Alegre, RS, Brazil) at 37ºC for 48 hours, and S. mutans was placed in a microaerophilic environment in an anaerobic jar (Permution, Curitiba, PR, Brazil).Microorganisms were identified by evaluating the macroscopic morphology of the colonies in differential selective culture media.OHRQoL was evaluated by the Brazilian version of the Oral Health Impact Profile in Edentulous Adults by OHIP-EDENT inventory. 23This instrument comprises 19 items that can be answered on a 3-point Likert scale and provides a summary score ranging from 0 to 38.Lower scores represent a better OHRQoL, which has been conceptually defined as the low impact of oral conditions on oral functions and wellbeing.Items in the translated version allowed for grouping into 4 subscales, representing different domains or dimensions of the perceived impact, such as masticatory-related complaints or psychological disability and discomfort.

Figure 2 .Figure 1 -
Figure 2. Staphylococcus spp.may have a low positive correlation with discomfort and chewing disability showed a negative correlation with salivary flow, and this factor had a negative correlation with DS severity, different to what was reported by Lucena, et al. 27 (2010).
Therefore, well-established management incorporated into daily practices related to oral and denture hygiene may be essential to control and prevent DS.In this scenario, future controlled clinical studies could compare individuals before and after DS treatment, including in other populations such as younger patients, to highlight the relationship between microbial load and DS.When studies specifically evaluated the biofilm of gram-positive bacteria, such as Staphylococcus spp., it was observed that their adherence to surfaces exhibited different behaviors compared to C. albicans.Regarding biofilm formation, studies have shown thatC.albicans can form a more structured and complex biofilm when compared with S. aureus, presenting better adherence and a more homogeneous biofilmlike structure, which seems to adhere mainly as individual or grouped cocci, and biofilm-like structures attached to the different morphological forms of the fungus.32The microbial count of Staphylococcus spp.had a negative correlation with the degree of DS, which can be explained by the lower adhesion of these microorganisms to the surfaces evaluated.32

Figure 2 -
Figure 2-Correlation between OHRQoL, count of Staphylococcus spp., and enterobacteria on the mucosal palate Each point corresponds to the mean rank of correlation between colony-forming units of microorganisms and domains of quality of life.

Finally
, one significant limitation is that the sample size was an estimate based only on the colony-forming CAMPOS EN, CLEMENTE LM, PIZZIOLO PG, OLIVEIRA VC, MACEDO AP, WATANABE E, SILVA-LOVATO CH, RIBEIRO AB unit (CFU) counts, information commonly presented in previous studies.However, many risk factors for DS should be investigated by controlled studies.To this end, the results of this study can contribute to future sample estimates.ConclusionBased on the findings of this clinical study, the main risk factors for developing denture stomatitis are:1.Being female, consuming alcoholic beverages as a young adult (18-27 years), and the presence of Candida spp., Candida albicans, and Candida glabrata on the dentures and palate.

2 .
The quality of life, especially domains of OHIP-EDENT D2, D3, and D4 may be slightly influenced by the CFU count of Staphylococcus spp., and domains D3 and D4 by enterobacteria.This relationship appears to be potentially significant and should be investigated as a trend in randomized clinical trials.

Table 1 -
Sociodemographic data of the sample expressed in absolute numbers and correlation to DS CAMPOS EN, CLEMENTE LM, PIZZIOLO PG, OLIVEIRA VC, MACEDO AP, WATANABE E, SILVA-LOVATO CH, RIBEIRO AB Kendall's tau_b test (p<0.05)*.DS: Denture Stomatitis.0: healthy mucosa; IA: petechiae in normal palatal tissue; IB: localized area of inflammation of the denture-bearing area; II: generalized area of inflammation of the denture-bearing area; III: hyperplasic palatal surface with inflammations of the denture-bearing area.J Appl Oral Sci.2023;31:e20230192 6/13 27 years) was a determining factor for the positive correlation with Kendall's tau_b test (p<0.05)*.DS: Denture Stomatitis.LS 1. Have you ever smoked 100 cigarettes in your life?LS 2. How old were you when you started smoking?LS 3. How many years did you smoke?LS 4. Do you use alcoholic beverages?LS 5. How old were you when you started using alcohol?0, IA, IB, II, III are degrees of DRS according to the Newton Modified Classification.Being, 0: healthy mucosa; IA: petechiae in normal palatal tissue; IB: localized area of inflammation of the denture-bearing area; II: generalized area of inflammation of the denture-bearing area; III: hyperplasic palatal surface with inflammations of the denture-bearing area.

Table 2 -
Correlation of lifestyle and degree of DSRelation between the risk factors for the severity of denture stomatitis and quality of life of complete edentulous individuals: a cross-sectional study *. DS: Denture Stomatitis.SF: Salivary flow.Equal letters indicate statistical equality.0, IA, IB, II, III are degrees of DRS according to the Newton Modified Classification.Being, 0: healthy mucosa; IA: petechiae in normal palatal tissue; IB: localized area of inflammation of the denture-bearing area; II: generalized area of inflammation of the denture-bearing area; III: hyperplasic palatal surface with inflammations of the denture-bearing area.

Table 3 -
Descriptive and inferential analysis of the use of medicaments, salivary flow related to degree of DS, and use of medicaments related to salivary flow CAMPOS EN, CLEMENTE LM, PIZZIOLO PG, OLIVEIRA VC, MACEDO AP, WATANABE E, SILVA-LOVATO CH, RIBEIRO AB Palate.CI: Confidence Interval.NV: There was no variation.Equal letters indicate statistical equality.0, IA, IB, II, III are degrees of DRS according to the Newton Modified Classification.Being, 0: healthy mucosa; IA: petechiae in normal palatal tissue; IB: localized area of inflammation of the denture-bearing area; II: generalized area of inflammation of the denture-bearing area; III: hyperplasic palatal surface with inflammations of the denture-bearing area.

Table 5 -
Descriptive and inferential analyses of the CFU + 1 count (Log 10) of Candida spp.; C. albicans, C. tropicalis; C. glabrata, other species of Candida, enterobacteria, Staphylococcus spp., and Streptococcus mutans on the denture, and correlation to the degree of DS CAMPOS EN, CLEMENTE LM, PIZZIOLO PG, OLIVEIRA VC, MACEDO AP, WATANABE E, SILVA-LOVATO CH, RIBEIRO AB